Viruses
A seasonal influenza A H1N1 virus (A/Oklahoma/447/08), a pandemic H1N1/09 virus (A/Hong Kong/415742/09, H1N1pdm) and two HPAI H5N1 viruses isolated from patients with fatal human H5N1 disease in Hong Kong in 1997 (A/Hong Kong/483/97) and in Vietnam in 2004 (A/Vietnam/1203/04) were used. Viruses were isolated and cultured in Madin-Darby canine kidney (MDCK) cells. Virus titration was done using tissue culture infection dose 50% (TCID50) assay. Infections were all performed in the BSL-3 bio-containment facility at the Core Facility of Li Ka Shing Faculty of Medicine, HKU.
Viral Titration by TCID50 assay
MDCK cells were seeded on 96-well tissue culture plates one day before the viral titration assay. Cells were washed once with PBS and changed to serum-free MEM medium with 1% PS and L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin. Virus samples or culture supernatants were titrated in serial half-log10 dilutions with serum-free medium prior to the addition of the diluted virus to MDCK cell plates in quadruplicate. The highest viral dilution leading to cytopathic effect (CPE) in ~50% of inoculated wells was estimated using the Karber method.
Primary human Nϕs isolation and infection
10ml of peripheral blood was drawn from healthy volunteers aged from 24 to 40 years old and CGD patients aged from 5 to 16 years old. Informed consent was sought and the collection of patients’ peripheral blood was approved by the HKU/HA HKW Institutional Review Board (UW 10-430 and 15-026).
Primary human Nϕs were isolated by density centrifugation with Histopaque 1077 and 1119. The granulocyte layer was purified from red blood cell using lysis buffer and washed with PBS as described (1). The purified Nϕs were cultured in RPMI-1640 medium without phenol red with 10% FBS and 1% PS. The average yield of this protocol was 1.57 ± 0.21×106 cell/ml, n = 5 with an approximately 20-21 hours(h) lifespan. The freshly isolated Nϕs were seeded at a density of 1×105 cell per well onto a 24-well tissue culture plates and settled for 1h before infection experiments. Thereafter, influenza virus at a multiplicity of infection (MOI) of 0.01 and 2 was added into the Nϕ cultures. For transepithelial migration experiments, 5×105 naïve Nϕs were directly added to the apical chamber with alveolar epithelial cells as described below.
Isolation and culture of primary human type I-like pneumocytes (Pϕ)
Primary human type I-like Pϕs were isolated from the lung tissue obtained from patients underwent lung resection in the Department of Cardiothoracic Surgery, Queen Mary Hospital, Hong Kong SAR, with the approval of Institutional Review Board of the University of Hong Kong and Hospital Authority Hong Kong West Cluster (UW 10-430). Pϕs were prepared as previously described (22). Briefly, lung tissue was chopped into pieces of 0.5mm thickness using tissue chopper and digested using a combination of trypsin and elastase for 45 min at 37°C in a shaking water-bath. The cell population was purified by cell attachment, percoll density gradient centrifugation and magnetic cell sorting. The cells were maintained in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions, and small airway growth medium, SAGM, (Lonza, USA) was used and changed every 48h starting from 48 h after cell plating.
Neutrophil Transepithelial migration assay
3×105 Pϕs were seeded on the basolateral side of a transwell insert with a 3.0µm pore size membrane by inverting the transwells (Figure 1A). The setup was incubated at a 37°C, 5% CO2 incubator for 6 h to allow cell attachment. After 6 h, the transwells were flipped upright and medium was added back to both apical and basolateral chambers and the monolayer was allowed to grow for at least 48 h into a tight monolayer, confirmed by transepithelial resistance measurements.
To infect the Pϕs, transwells were inverted with the apical surface of the Pϕs exposed to 80ml of influenza virus at a MOI of 0.01 for 1 h followed by PBS wash for three times. The transwells were then returned to its original orientation in a 24 well ultra-low adhesion plate with small airway growth medium (SAGM, Lonza). At 6 and 24 hpi, 5×105 naïve Nϕs were added to the apical chamber, and 90 mins were allowed for the transepithelial migration to take place (23). After 90 mins, the number of Nϕs that migrated though the epithelial monolayer into the lower chamber was counted and expressed in percentage of transmigration.
Influenza viral gene, protein expression and infectious titer determination
Virus transcription was assessed by influenza matrix (M) gene copy numbers in RNA lysate of the infected cells at 1, 3, 6 and 16 hpi. Infected Nϕs on coverslips were fixed with 4% paraformaldehyde (PFA) at 6 hpi for immunofluorescence assay using FITC-conjugated antibodies against influenza nucleoprotein (NP) and M protein. The supernatant samples of the infected cultures were collected at 1, 6 and 16 hpi for viral titration assay in MDCK cell in 96 well-plate format and expressed in TCID50/ml.
Immunofluorescence staining for influenza antigen
4% PFA fixed Nϕs were stained with mouse anti-influenza virus NP and M protein antibodies conjugated with fluorescein isothiocyanate (FITC) (Dako Diagnostics Ltd., Ely, United Kingdom). After staining, the coverslips were mounted onto glass slides using a mounting medium with 4’, 6-diamidino-2-phenylindole (DAPI) (Vectashield mounting medium with DAPI; Vector Laboratories Inc.).
Thermal inactivation of virus
In order to have an objective measure of the overall viral load in the experimental setup, including those infectious viruses that were thermally inactivated at 37°C during the experiment period, thermal inactivation measurements of influenza virus of the two subtypes at a gradient of concentrations was performed. 1 ml of viruses at different concentrations (102 – 106 TCID50/ml) was put in a 24-well plate and placed in the 37°C incubator. 130 ul of the virus were collected at 1, 6, and 16 h post incubation along the experimental duration of our in vitro study. The virus titer of these samples was measured by the viral titration and thus the thermal inactivation curves of these viruses at 37°C were constructed (Figure 2C, grey dotted line). These curves were used to plot along with the viral titer of the virus inoculated Nϕ culture collected at the same time point. The difference between the titer measured in the Nϕ supernatant and the thermal inactivated control indicated the actual viral load produced by the infected cells.
Cytokine and chemokine gene expression in naïve neutrophils after influenza virus infection
At 6 and 16 hpi, the Nϕs infected with different strains of influenza viruses were lysed using RLT and beta-mercaptoethanol and mRNA of cells were extracted using QIAGEN RNAeasy Kit following manufacturer’s instruction. Reverse transcription was carried out with a PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China). The quantitative PCR (qPCR) SYBR Premix Ex Taq (Takara Biotechnology, Dalian, China) was used and the real-time qPCR assays were run on a ViiATM 7 Real Time PCR System (Life technology, Carlsbad, CA). Expression of these genes was normalized by using the product of the β-actin housekeeping gene mRNA. Viral M gene expression, host cytokines (TNF-α, IFN-β) and chemokines (CXCL10, CCL2, IL-8 and MIP-1 α) gene expression and the housekeeping (β-actin) gene expression in absolute copy numbers were determined using a standard curve generated by a serial dilution of a standard plasmid with known copy numbers, which was included in the qPCR simultaneously. These gene expressions were normalized by using the housekeeping gene product β-actin mRNA and expressed as copy number per 105 β-actin expressions.
Measurement of NET formation
NET formation was visualized with immunofluorescence staining with 5μM of Sytox Green (Invitrogen, California, United States) before fixation 4% PFA. Coverslips were mounted onto glass slides with VECTASHIELD ® Antifade Mounting Medium with DAPI.
To quantitatively assess the level of NET formation induced by the different strains of influenza virus, Nϕs (1×104 cell/well) were seeded into a sterile 96-well flat-bottom black plate. At 6 hpi, the cells were stained with 5μM Sytox Green for 5 mins and relative fluorescence intensity was determined using a FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany) at an excitation wavelength of 485nm and an emission wavelength of 520nm. The relative fluorescence units (RFU) of infected cells were obtained after subtracting the fluorescence of unstimulated neutrophils. The RFU of the mock-infected Nϕs was set as 100%. The plotted RFU of Nϕs infected with influenza viruses was calculated by dividing the raw RFU reading by that of mock-infected Nϕs.
Scanning electron microscopy (SEM)
To observe the formation of NET, control and infected Nϕs were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate-HCL buffer pH 7.4 at 1, 6 and 21hpi. Fixed cells on coverslip were washed in cacodylate buffer with 0.1M sucrose to remove excess fixative and post-fix in osmium tetroxide for 1h at room temperature. The cells were then dehydrated using increased concentration of ethanol each for 15 mins from 30%, 50%, 70%, 90%, to 100%. The cells were dried in a critical point dryer using liquid carbon dioxide as transitional fluid and the coverslips were then mount on the specimen holders. A thin layer (100-200Å) of metallic film was coated on the specimen surface using a vacuum evaporator. The samples were kept in a desiccator until viewing. Hitachi S-4800 FEG scanning electron microscope (Electron Microscope Unit, HKU) was used to examine these samples, with the EMAX energy software.
Statistical analysis
Mock-infected cells served as negative controls and each experiment was repeated at least three times using cells from different donors and the dataset were pooled. The mean and standard error of mean (SEM) of three experiments were shown. The gene expression of different cytokines and chemokines was compared among different treatments at each time point using one-way ANOVA. P value less than 0.05 was deemed to be statistically significant. Bonferroni multiple comparison test was used as the post test to determine significance between treatments. Results were considered significant at p ≤ 0.05. Statistical analyses were done with GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA).